Ontogenesis of alpha- and beta-receptors located on cerebral microvessels

Adrenergic receptors in rat brain microvessels were studied during ontogenesis. Microvessels were prepared by albumin floatation and glass bead filtration techniques from cortices of 10, 20- and 90-day-old rats. The lower level of alpha 1- alpha 2- and beta-receptor sites observed in early life may correlate with the lower capacity of cerebral vascular regulatory mechanism in this period.     

Protein and RNA synthesis in cerebral microvessels: A radioautographic study

The distribution of protein and ribonucleic acid (RNA) synthesized by telencephalon slices was determined by radioautography at the light microscope level. Previous studies had shown that, in telencephalon slices, the cells associated with the cerebral microvasculature were synthesizing protein at very high rates compared to other cerebral cells such as neurons. In this study, therefore, particular emphasis was placed on the distribution of newly synthesized protein and RNA among the various cell types of the microvasculature. Rat telecephalon slices were incubated with either [³H]leucine or [³H]uridine for 1 h. The³H-protein that was found in association with microvessels was present in endothelial cells, pericytes and smooth muscle cells. There was also extensive labeling of cellular processes adjacent to the abluminal side of the basement membrane and extending as far as 15 μm from the lumen. The latter processes may have been astrocyte glial projections. All [³H]RNA found associated with microvessels was, however, restricted to endothelial cells and pericytes. This data is discussed in relation to the induction of a 71,000 dalton protein which occurs within minutes of the preparation of tissue slices, and requires the synthesis of new RNA.     

Effects of oxysophoridine on amino acids after cerebral ischemic injury in mice

Background:

Our previous studies demonstrated that oxysophoridine (OSR) had neuroprotective effects on mice through antioxidant and anti-apoptotic mechanisms. In this study, we investigated whether OSR could influence the release of amino acids in ischemic mice brains.

Materials and Methods:

Male ICR mice were scheduled to undergo 2 h middle cerebral artery occlusion (MCAO) and 24 h reperfusion. Before MCAO, mice in corresponding groups were intraperitoneally injected with OSR (62.5, 125 and 250 mg/kg) for seven successive days. After reperfusion, neurological scores were estimated, infarct volume and the brain water content were assessed. The levels of glutamate (Glu), aspartate (Asp), γ-aminobutyric acid (GABA) and Glycine (Gly) were measured by amino acid analyzer.

Results:

OSR significantly decreased neurological scores, reduced infarct volume and the brain water content. After treatment with OSR of 250 mg/kg, the contents of Glu, Asp, GABA and Gly in mice brains could maintain at a normal level compared with MCAO group mice. The Glu/GABA ratio was significantly decreased in OSR group mice.

Conclusion:

These findings indicate that OSR has a protective effect on cerebral ischemic injury and helps to maintain the amino acids homeostasis after reperfusion for a long time.     

Ontogenesis of α- and β-receptors located on cerebral microvessels

Adrenergic receptors in rat brain microvessels were studied during ontogenesis. Microvessels were prepared by albumin floatation and glass bead filtration techniques from cortices of 10-, 20- and 90-day-old rats.The lower level of α1- α2- and β-receptor sites observed in early life may correlate with the lower capacity of cerebral vascular regulatory mechanism in this period.     

Sexual differences in mitochondrial and related proteins in rat cerebral microvessels: A proteomic approach

Sex differences in mitochondrial numbers and function are present in large cerebral arteries, but it is unclear whether these differences extend to the microcirculation. We performed an assessment of mitochondria-related proteins in cerebral microvessels (MVs) isolated from young, male and female, Sprague-Dawley rats. MVs composed of arterioles, capillaries, and venules were isolated from the cerebrum and used to perform a 3 versus 3 quantitative, multiplexed proteomics experiment utilizing tandem mass tags (TMT), coupled with liquid chromatography/mass spectrometry (LC/MS). MS data and bioinformatic analyses were performed using Proteome Discoverer version 2.2 and Ingenuity Pathway Analysis. We identified a total of 1969 proteins, of which 1871 were quantified by TMT labels. Sixty-four proteins were expressed significantly (p < 0.05) higher in female samples compared with male samples. Females expressed more mitochondrial proteins involved in energy production, mitochondrial membrane structure, anti-oxidant enzyme proteins, and those involved in fatty acid oxidation. Conversely, males had higher expression levels of mitochondria-destructive proteins. Our findings reveal, for the first time, the full extent of sexual dimorphism in the mitochondrial metabolic protein profiles of MVs, which may contribute to sex-dependent cerebrovascular and neurological pathologies.     

Regulation of protein kinase C activity in cerebral microvessels

Regulation of protein kinase C (PKC)-mediated responses may occur by inhibition of PKC-dependent phosphorylation or by dephosphorylation of targets by specific phosphatases. Mechanisms for the regulation of PKC were examined in isolated cerebral microvessels and compared to those in brain. The data demonstrated that inhibitors of phosphorylation are responsible for the regulation in brain microvessels while dephosphorylation by protein phosphatases accounts for a substantial portion of the regulation of the PKC response in brain. In addition, the inhibitory activity apparently increases with age. These results suggest that the control of PKC may be cell-type specific and developmentally regulated.     

犬脑干持续缺血模型氨基酸含量的动态变化

目的 :探讨氨基酸递质及调质在缺血性脑干损伤中的作用。方法 :建立犬脑干缺血模型 ,测定脑干缺血 3 0min组 ,缺血 3h、6h、12h后氨基酸的含量。结果 :与假手术组相比 ,Glu ,Asp ,Gly ,GABA ,Tau ,Ser ,Gln ,Ala及Thr的含量均随缺血时间的延长而不断增加。海风藤预处理可使缺血时Glu ,Asp及Ser含量的增高程度显著降低 ,使Gly ,GABA及Tau的增高显著加强。结论 :脑干持续缺血后Glu ,Asp及Ser可能是脑干缺血损害的生化基础。海风藤在脑干缺血中起神经保护作用     

Somatostatin release from cerebral cortical cells: influence of amino acid neurotransmitters

We examined the ability of several putative amino acid neurotransmitters to influence immunoreactive somatostatin (IRS) release from cultured rat cerebral cortical cells. The cells were exposed to either or sequential incubations in various concentrations of glutamate (Glu), aspartate (Asp), GABA, glycine, taurine and arginine. Glu and Asp were stimulatory to IRS release, whereas GABA was inhibitory. Glu-induced IRS release was calcium-dependent. Glycine and taurine were weak stimulants.     

Subtypes of β-adrenergic receptors in rat cerebral microvessels

The¹²⁵I-labeled iodohydroxybenzylpindolol (IHYP) binding to β-receptors on brain microvessels is inhibited by isoproterenol, epinephrine and norepinephrine, with K_i values of 2 × 10⁻⁷M, 2.5 × 10⁻⁶M and 1.2 × 10⁻⁵M, respectively. A modified Scatchard analysis of the inhibitory effects of practolol, metroprolol and zinterol on IHYP binding has shown that the proportion of β₂-receptors in our preparation is about 80% of the total β-adrenergic receptor population. Our data indicate that the β-adrenergic receptors located on cerebral microvessels are of both β₁ and β₂ types, with a predominance of the β₂ type.     

Inhibitory and excitatory amino acids in the cerebrospinal fluid of children with two types of cerebral palsy*

BACKGROUND： Under normal conditions, excitatory amino acids are dynamically balanced with inhibitory amino acids. Excitatory amino acids have been implicated in perinatal brain injury. OBJECTIVE： To investigate differences in the levels of the excitatory amino acids glutamic acid and aspartic acid, and the inhibitory amino acid gamma-aminobutyric acid （GABA） in the cerebrospinal fluid （CSF） of children with spastic cerebral palsy or athetotic cerebral palsy. DESIGN, TIME AND SETTING： Case-control exploratory observation of neurotransmitter in patients. The experiment was performed in the Pediatrics Department of the Second Affiliated Hospital of Changsha Medical College, the Cerebral Palsy Center of Xiangtan Affiliated Hospital of South China University and the Pediatrics Department of Xiangya Hospital, between February 2006 and May 2007. PARTICIPANTS： We selected 27 children with cerebral palsy, including 13 with spastic cerebral palsy and 14 with athetotic cerebral palsy. We selected 10 patients who were not affected by any neurological disease as controls. METHODS： Two mL blood-free CSF was harvested between the third and fourth lumbar vertebrae of each patient after anesthesia, and stored at -70℃. One mL CSF was mixed with 10 mg sulfosalicylic acid and placed in ice-bath for 10 minutes, then centrifuged 2 000 g for 10 minutes. The supernatant was collected for amino acid quantitation. MAIN OUTCOME MEASURES： The concentrations of glutamic acid, aspartic acid and GABA in the CSF were determined by high-performance liquid chromatography and fluorometric method. The correlation of glutamic acid, aspartic acid and GABA levels with muscular tension in children with cerebral palsy was analyzed using linear dependence. RESULTS： The concentration of GABA was significantly lower in both spastic cerebral palsy and athetotic cerebral palsy patients than in the control group （P 〈 0.01）. Glutamic acid and aspartic acid were significantly higher in both cerebral palsy groups than in the control     

The effect of in vitro and in vivo lead intoxication on monosaccharide transport in isolated rat brain microvessels

The effect of lead on facilitated transport of monosaccharides into isolated brain microvessels was investigated. Preparations from control rats and from rats exposed to lead acetate at various doses (0.025–1.0 mg lead/g body wt/day) from days 5–25 postnatally were studied. The time courses of uptake for various concentrations of 3-O-methylglucose (3OMG) were determined. Values of 0.6 min and 2.0 min for 5 and 50 mM 3OMG, respectively, were obtained in the case of preparations from control animals. In contrast, the half-time of uptake was about 1 min for both sugar concentrations for microvessels isolated from animals treated at 1.0 mg lead/g body wt/day. This indicates both a block in facilitated transport of the sugar, as well as damage to the endothelial cells resulting in increased passive permeability. There was a dose-response relationship for the effect of in vivo lead treatment on 3OMG transport in isolated microvessels. Microvessel preparations from younger animals (treated at 1.0 mg/g body wt for two days prior to sacrifice at 7 days postnatally) were more sensitive to lead treatment than preparations from adults; equivalent lead burden induced a greater increase in passive permeability to 3OMG in the younger animals.In other experiments microvessels isolated from untreated rats were preincubated with lead acetate. A concentration of 0.1 μM lead acetate in vitro abolished facilitated transport of 3OMG.     

dl-3n-butylphthalide prevents stroke via improvement of cerebral microvessels in RHRSP

The purpose of the study is to establish a model of cold-induced stroke in hypertensive rats, and to study the preventive effect of dl-3n-butylphthalide ( NBP ) on stroke. Stroke-prone renovascular hypertension(RHRSP) was created in Sprague-Dawley rats. The animals were assigned randomly to NBP, aspirin treated and vehicle control group, with administration of the medications for 7 days, and then subjected to cold treatment in an environmentally controlled chamber for 3 days to induce the occurrence of stroke. The incidence of stroke, the volume of the brain lesion, patency of the microvessels by FITC-dextran perfusion and the number of microvessels by immunohisochemical detection of vwF were investigated. Cold induced different types of stroke in RHRSP. The incidence of ischemic stroke and the volume of the infarct were decreased, and the perfused microvessels were increased with NBP pretreatment. Our data suggest that NBP prevents cold-induced ischemic stroke via improvement of cerebral microvessels.     

Behavioural and histopathological alterations in mice with cerebral malaria

Different features of sensorimotor function and behaviour were studied in murine cerebral malaria (CM) and malaria without cerebral involvement (non-CM) applying the primary screen of the SHIRPA protocol. Histopathological analysis of distinct brain regions was performed and the relative size of haemorrhages and plugging of blood cells to brain vasculature was analysed. Animals suffering from CM develop a wide range of behavioural and functional alterations in the progressive course of the disease with a statistically significant impairment in all functional categories assessed 36 h prior to death when compared with control animals. Early functional indicators of cerebral phenotype are impairments in reflex and sensory system and in neuropsychiatric state. Deterioration in function is paralleled by the degree of histopathological changes with a statistically significant correlation between the SHIRPA score of CM animals and the mean size of brain haemorrhage. Furthermore, image analysis yielded that the relative area of the brain lesions was significantly larger in the forebrain and brainstem compared with the other regions of interest. Our results indicate that assessment of sensory and motor tasks by the SHIRPA primary screen is appropriate for the early in vivo discrimination of cerebral involvement in experimental murine malaria. Our findings also suggest a correlation between the degree of functional impairment and the size of the brain lesions as indicated by parenchymal haemorrhage. Applying the SHIRPA protocol in the functional characterization of animals suffering from CM might prove useful in the preclinical assessment of new antimalarial and potential neuroprotective therapies.     

Amino acid transmitter candidates in various regions of the primary olfactory cortex following bulbectomy

The effect of bulbectomy on amino acid levels in 5 regions of the rat primary olfactory cortex has been monitored. Glutamate levels were significantly lowered in the lateral olfactory tract only. In contrast, aspartate levels were significantly reduced in all regions except the periamygdaloid cortex. The results suggest transmitter heterogeneity of the tract fibres and confirm a likely transmitter role for aspartate for some tract terminals.     

不同脑温状态下地西泮对大鼠脑缺血组织氨基酸及自由基的影响

目的 探讨不同脑温状态下地西泮对大鼠脑缺血组织谷氨酸(Glu)、γ-氨基丁酸(GABA)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)及丙二醛(MDA)的影响.方法 建立大鼠大脑中动脉缺血再灌注模型,诱导目标脑温,测定轻度高温、常温、亚低温状态下各组脑缺血组织Glu、GABA、SOD、GSH-Px及MDA含量.结果 (1)与常温假手术组比较,常温脑缺血对照组及常温地西泮组Glu、MDA水平显著增高(均P<0.001),SOD、GSH-Px水平显著降低(均P<0.001),GABA在常温脑缺血对照组差异无统计学意义(P>0.05),在常温地西泮组则显著增高(P<0.01).(2)与常温脑缺血对照组比较,常温地西泮组GSH-Px显著增高(P<0.001),MDA显著降低(P<0.001),而Glu、GABA、SOD差异均无统计学意义(均P>0.05).(3)与常温地西泮组比较,轻度高温地西泮组Glu、MDA显著增高(均P<0.001),SOD、GSH-Px显著降低(均P<0.001),GABA差异无统计学意义(P>0.05);亚低温地西泮组Glu、MDA显著降低(P<0.01～0.001),GABA、SOD、GSH-Px显著增高(均P<0.001).结论 亚低温状态下,地西泮显著上调GABA水平,有利于地西泮"抑制性保护"机制的建立,从而增强地西泮的神经保护作用.     

急性脑梗死患者脑脊液兴奋性氨基酸水平的变化及其临床意义

目的 研究急性脑梗死患者脑脊液（CSF）兴奋性氨基酸含量变化及其临床意义。方法 采用高效液相层析方法测定30例急性脑梗死患者及20例偏头痛患者（对照组）CSF谷氨酸（Glu）、天门冬氨酸（Asp）的含量。结果 脑梗死组患者CSF中Glu、Asp含量显著高于对照组（均P〈0．001）。脑梗死组患者CSF中Glu和Asp含量与梗死体积呈显著正相关（r=0．75，P〈0．01；r=0.437，P〈0．05），CSF中Clu含星与神经功能缺损程度亦呈正相关（r=0．55，P〈0．01）。结论 脑梗死发生后CSF中Glu、Asp含量明显升高提示其参与了脑梗死的病理生理过程CSP中Glu、Asp含量可反映脑梗死患者的病情及梗死灶的大小。     

Adrenergic,serotoninergic, histaminergic,and imipramine binding sites in post-mortal human cerebral microvessel preparations

Summary Cerebral microvessels were prepared from fresh and frozen human brain samples obtained from autopsy cases. Structural integrity and purity of the microvessels were confirmed by light and electron microscopy, and by measurement of the enzymatic marker -glutamyltranspeptidase. Similar morphological and enzymatic characteristics were found for the microvessels prepared from fresh and frozen brain samples. Radioligand binding experiments indicated the presence both in the fresh and frozen microvessel preparations of specific alpha₁-, alpha₂-, and beta-adrenergic, histamine H₁, serotonin S₁ and imipramine binding sites, although the density of beta-adrenergic and histamine h₁ specific binding sites were lower in the frozen samples than in the fresh samples. Low levels of specific binding to muscarinic, GABAergic and serotonin S₂ sites (with respect to the specific binding densities in the crude homogenates) were found in the microvessel preparations.     

Amino acid concentrations in cerebrospinal fluid and plasma in Alzheimer's disease and healthy control subjects

Summary Cerebrospinal fluid (CSF) and plasma levels of 18 amino acids were studied in 22 subjects with dementia of the Alzheimer type (DAT) and in 11 healthy volunteers with no clinical or family history of dementia. Significant decreases of plasma taurine and glutamate were seen in the DAT case compared with the controls. The CSF concentrations of glycine, leucine and valine were also significantly reduced in the DAT cases. Furthermore, in the DAT cases significant decreases were observed in the ratio between CSF and plasma (CSF/P) levels for alanine, glutamine, glycine, phenylalanine and valine, when compared with controls. In the DAT group there were significant correlations between behaviour and CSF glutamine; memory and cognitive functions and CSF valine; copying ability and CSF glutamate. CSF/P ratios of glutamine and glutamate correlated with behaviour and copying performances, respectively. The results of this study provide further evidence for a disruption of amino acid metabolism in DAT.     

Amino acid neurotransmitter levels in gliomas and their relationship to the incidence of epilepsy

The cause of epilepsy due to gliomas is not known. Explanations that it is due to mass effect, infiltration and site of the tumour appear insufficient. We have investigated the possibility that epilepsy due to gliomas is caused by interference with normal GABA and glutamate uptake and metabolism in the surrounding cortex. Analysis of human glioma biopsy specimens for the amino acid neurotransmitters and glutamine has shown that gliomas associated with epilepsy have a higher concentration of glutamine. This may be of importance since an elevated concentration of glutamine has been shown to be associated with the onset and severity of cobalt-induced epilepsy.